Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry


Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leukocyte antigens (HLA) on the surface of native tumor tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumor tissue samples harboring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumor-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient`s tumor and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumor material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

Sample Processing Protocol

Tumor tissue samples from 25 melanoma patients (Supplementary Table 1 and 2) were used for analysis of biochemically purified HLA class I and II binding peptides. In total, we performed 140 MS measurements of purified peptides by LC-MS/MS analysis using a state-of-the-art mass spectrometer, followed by stringent bio(informatics) analyses in the MaxQuant environment.

Data Processing Protocol

We employed the MaxQuant computational proteomics platform version Andromeda, a probabilistic search engine incorporated in the MaxQuant framework, was used to search the peak lists against the UniProt databases (Human 85,919 entries, Sep 2014), and a file containing 247 frequently observed contaminants. For identification of mutated peptide ligands, customized references databases were used. N-terminal acetylation (42.010565 Da), methionine oxidation (15.994915 Da) and phosphorylation (79.9663304 Da on serine, threonine and tyrosine) were set as variable modifications. The second peptide identification option in Andromeda was enabled. The enzyme specificity was set as unspecific. A false discovery rate of 0.01 was required for peptides for the global ligandome analysis and for the phospho-HLA peptides identification. We applied in addition a less stringent threshold of 5% for the identification of mutated peptide ligands. No protein false discovery rate nor permutation rules were set in MaxQuant in creating the decoy database. The initial allowed mass deviation of the precursor ion was set to 6 ppm and the maximum fragment mass deviation was set to 20 ppm. We enabled the ‘match between runs’ option, which allows matching of identifications across different replicates that belongs the same patient, in a time window of 0.5 min and an initial alignment time window of 20 min.


Michal Bassani-Sternberg, UNIL/CHUV
Michal Bassani-Sternberg, Head / Immunopeptidomics unit Department of Oncology UNIL/CHUV Ludwig Cancer Research Center Biopole III, Chemin des Boversses 155 Epalinges, 1066 Switzerland ( lab head )

Submission Date


Publication Date





Q Exactive


Not available


phosphorylated residue


Not available

Experiment Type

Shotgun proteomics


    Bassani-Sternberg M, Bräunlein E, Klar R, Engleitner T, Sinitcyn P, Audehm S, Straub M, Weber J, Slotta-Huspenina J, Specht K, Martignoni ME, Werner A, Hein R, H Busch D, Peschel C, Rad R, Cox J, Mann M, Krackhardt AM. Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry. Nat Commun. 2016 Nov 21;7:13404 PubMed: 27869121